Synaptic dysfunction and A accumulation are the key features of Alzheimer's disease (AD) pathology. Numerous studies have shown that A can be produced in activity-dependent manner, and this correlates with synaptic vesicle exocytosis. PS1/g-secretase is present at the synaptic terminals, and our preliminary data show that it changes its conformation rapidly and reversibly in concert with synaptic activity and calcium (Ca2+) influx. Presenilin 1 (PS1) is the catalytic component of ?-secretase, which liberates the C-terminus of A peptide, and thus determines both amount of A and which A species will be produced: A40 or the longer, highly fibrillogenic and neurotoxic A?42. However, the cell biological mechanisms that control precision of the PS1/?-secretase cleavage site and local A production at the synapse remains unknown. PS1 phosphorylation by several kinases has been reported; however mechanistic effect of the PS1 phosphorylation remains unclear. Aim 1 will determine whether neuronal activity/Ca2+-induced phosphorylation of PS1/?-secretase represents the regulatory mechanism of PS1 conformation and function at the synapse. Furthermore, our recent proteomics screen of mouse brain lysates in the presence or absence of calcium identified two novel PS1 interacting proteins, synapsin1 (Syn1) and synaptotagmin1 (Syt1), that showed strong but opposing Ca2+-dependent profiles of binding to PS1. Syn1 anchors synaptic vesicles to actin filaments, but releases them after Ca2+-induced Syn1 phosphorylation. Syt1 acts as Ca2+sensor in neurotransmitter release. Aim 2 will explore whether, Ca2+ influx may function as a switch controlling PS1 conformation and interactions with Syn1 and Syt1. In addition, we will test if PS1 interactions with Syn1 and Syt1 modulate PS1/?-secretase and APP processing at the synapse in an activity-controlled manner. Aim 3 will validate physiological relevance of the newly found PS1 interaction with synaptic proteins in vivo by establishing if it is affected in aged and/or diseased brain, and whether these interactions can be manipulated pharmacologically. This study will provide mechanistic data for PS1 conformational changes, will explore novel PS1 interactions with synaptic vesicle machinery proteins, and will elucidate novel A-dependent and independent role of PS1 at the synapse. Understanding these issues is of high importance because manipulation of the PS1 conformation and synaptic interactions may translate into novel therapeutic strategies.